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Chapter 14 Forensic DNA Typing Objectives • Students should gain an understanding of: – The use of the polymerase chain reaction (PCR) to make many copies of a DNA sequence – Short tandem repeats (STRs) and their forensic importance – The use of electrophoresis to analyze STRs – The Combined DNA Index System (CODIS) – DNA paternity testing – Mitochondrial DNA testing Introduction • The DNA in all cells of an individual is the same through the body. • DNA contains repeated sequences of genetic codes with core sequences that are unique to particular individuals. • The genetic code can be determined from a small amount of DNA. Restriction Fragment Length Polymorphisms (1 of 3) • DNA contains genes that control production of proteins in the body. • Other sections act as spacers between the coding areas. – The sequences of bases in the noncoding regions are used for DNA profiling. – The sequences vary greatly from person to person. Restriction Fragment Length Polymorphisms (2 of 3) • RFLP allows for the individualization of DNA evidence – Step 1: DNA is extracted from a chromosome – Step 2: restriction enzymes cut the DNA strands into fragments at specific base sequences Restriction Fragment Length Polymorphisms (3 of 3) • Disadvantages of RFLP – Takes 6–8 weeks to obtain results – Requires a large sample of intact, nondegraded DNA – Not amenable to high-volume sample processing – Produces very large DNA strands that are often damaged when the recovered DNA is partially degraded Polymerase Chain Reaction: A DNA Copy Machine (1 of 3) • Advantages of PCR – Allows many copies of a portion of DNA sequence to be manufactured in the DNA lab – Amplifies only those DNA regions that are of interest – Is fast and extremely sensitive Polymerase Chain Reaction: A DNA Copy Machine (2 of 3) • Thermocycling: DNA is repeatedly heated and cooled – 194 °F: the two complementary DNA strands separate – 140 °F: each primer finds and binds to its complementary sequence on the DNA strand – 162 °F: the DNA polymerase enzyme adds bases to extend the primer and to build a DNA strand that is complementary to the sample DNA Polymerase Chain Reaction: A DNA Copy Machine (3 of 3) • The thermocycling process is repeated to make more copies. • Each heating–cooling cycle doubles the number of copies. • DNA laboratory and technicians must take extreme care to eliminate extraneous DNA from the PCR amplification area. Short Tandem Repeats • STRs: locations on the sample DNA that contain a short sequence of bases that is repeated over and over – Four-base repeats are typically used for forensic purposes. – STRs are often recovered from bodies or stains that have started to decompose. – They can be amplified very quickly. DNA Sequence Variations among Individuals • Individuals differ genetically because they possess different combinations of alleles at numerous locations in their genomes. • Only 3% of a person’s DNA is involved in coding for proteins. • Mutations in noncoding regions have no effect on the phenotype of a person. • Loci selected for DNA typing are selectively neutral; they confer neither benefit nor harm to the individual’s ability to reproduce. Inheritance of Alleles • Alleles are inherited from an individual’s parents following the fundamental rules of genetics. • Different individuals posses different alleles in numerous loci in their genomes. • Investigators measure the length of STRs at different locations to determine a person’s genetic identity. Analyzing the STR by Electrophoresis (1 of 3) • Electrophoresis – Causes ions in solution to migrate under the influence of an electric field – Separates STRs according to their length: smaller DNA molecules move faster – Establishes the number of repeats and elucidates the genotype of the individual at each amplified locus Analyzing the STR by Electrophoresis (2 of 3) • Gel electrophoresis – After voltage is applied 2–3 hours, electrophoresis stops and the DNA is made visible. – Each group of similar-length molecules appears as a narrow band in the gel. – By comparing the locations of the bands in each sample lane to the ladder, the technician can determine the STR type for each sample. – Gel electrophoresis is slow, is difficult to automate, and can be dangerous. Analyzing the STR by Electrophoresis (3 of 3) • Capillary electrophoresis – Allows for greater heating than is possible with a slab gel – Uses a higher voltage, so molecules migrate much faster – Produces high-speed, high-resolution separations on extremely small samples – Uses laser fluorescence: fluorescent dye is attached to the PCR primer that amplifies the STR region of interest Multiplex DNA Analysis (1 of 4) • Multiple STR loci may undergo PCR amplification and be analyzed simultaneously. • Multiplexing is accomplished by placing each of the four dyes on specific primers and by adjusting the size of the STR amplicon produced. Multiplex DNA Analysis (2 of 4) • Multiplexing by size – Amplicons from different loci that are different sizes are clearly separated from one another and appear at different locations on the x-axis in the CE analysis. – It isn’t possible to multiplex more than five or six loci. Multiplex DNA Analysis (3 of 4) • Multiplexing by dye color – Different dyes amplify STR loci that are the same size and cannot be separated using CE. Multiplex DNA Analysis (4 of 4) • Multiplexing with multiple capillaries: capillary array electrophoresis – Parallel capillaries lie next to one another and process multiple samples simultaneously – Technique reduces the time between when a DNA sample is collected at the crime scene and when it is actually analyzed Forensic STRs • Most DNA databases rely on 10 or more STR loci, each of which is found on a different chromosome. • Standard nomenclature is used to designate the location of a DNA marker. – If the marker is part of a gene or falls within it, the gene name is used. – If the STR falls outside a gene region, its name indicates the chromosome and locus on which it is found. CODIS (1 of 4) • Combined DNA Index System – Created in 1994 as part of the DNA Identification Act – Consists of a national database containing the DNA of individuals convicted of sexual and violent crimes – Assisted in 20,000 investigations in 2004 CODIS (2 of 4) • Three tiers of CODIS – Local: labs maintain a local DNA index – State: combines the profiles of all local labs – National: compares profiles of all state systems CODIS (3 of 4) • All 50 states maintain databases for sexual offenders and convicted murderers • 49 states include violent felons • 43 states include all felons CODIS (4 of 4) • Use of CODIS – The computer compares the DNA profile submitted with profiles on file in the network. – If a match is found in the Convicted Offender Index, the lab is sent the identity of the perpetrator. – If a match is found in the Forensic Index, two crimes have been linked together. The labs must then verify the match; law enforcement may then pool resources to solve the crimes. Interpretation of DNA Profiles (1 of 3) • It is easier to use DNA to exclude a person from suspicion than to prove that the person is the only suspect. • The Innocence Project reports that three times more suspects are proven innocent by DNA analysis than are proven guilty. • The loci used for DNA matches must be chosen to minimize the chance that two people will have the same profile. Interpretation of DNA Profiles (2 of 3) • Hardy–Weinberg principle: allele frequencies remain constant from generation to generation and allele frequencies can be easily calculated – Frequency of a particular homozygote = allele frequency squared – Expected frequency for a heterozygote = 2 × the product of the two allele frequencies Interpretation of DNA Profiles (3 of 3) • Interpreting multiple DNA profiles: – Prevalence of a particular CODIS profile in the general population: multiply the genotype frequencies for all the loci together – Likelihood ratio: compares the probabilities of alternative events • The true discriminating power of CODIS is achieved by multiplying the individual frequencies of the 13 loci. Paternity Testing (1 of 2) • A child can receive only one of the father’s alleles and one of the mother’s alleles. • A familial pattern should be obvious by comparing the DNA profiles of mother, father, and child. Paternity Testing (2 of 2) • Paternity index: the likelihood that an allele from the child supports the assumption that the tested man is the true biological father • Combined paternity index: determined by multiplying the individual PIs for each locus tested Mitochondrial DNA Analysis (1 of 5) • Examination of recovered mitochondrial DNA is useful in circumstances of badly decomposed or burned bodies, old bones, and human hair without follicular tags • mtDNA is rarely used in criminal proceedings. • It has been useful for historical investigations. Mitochondrial DNA Analysis (2 of 5) • Mitochondrial DNA (mtDNA) – Is a circular DNA molecule that is only 16,569 pairs in circumference – Has no noncoding elements; every base has a function – For the most part, is the same in all individuals Mitochondrial DNA Analysis (3 of 5) • Variations in mtDNA – The D-loop contains two hypervariable regions whose sequences vary. – A difference of less than 3% is expected between unrelated individuals. Mitochondrial DNA Analysis (4 of 5) • DNA sequencing: determines the sequence of bases along a DNA strand • Anderson sequence: the first mtDNA hypervariable sequence to be determined; serves as a reference sample Mitochondrial DNA Analysis (5 of 5) • mtDNA is inherited from a person’s mother. • All brothers and sisters of the same mother have the same mtDNA, which is also the same as the mtDNA of their maternal grandmother and their mother’s siblings. The Y Chromosome: STRs and SNPs (1 of 2) • STR analysis is directed at the Y chromosome. • The Y chromosome contains polymorphisms that might eventually be used as forensic markers. The Y Chromosome: STRs and SNPs (2 of 2) • Single-nucleotide polymorphisms (SNPs) (2 of 2) – The base difference occurs at only one specific site. – SNPs at different loci can be determined simultaneously, producing an SNP DNA profile. Low-Copy-Number DNA Typing • Applications – Used when the quantifying test indicates too little DNA is available to perform a regular DNA analysis – Used only in cases where standard typing protocols have already failed • As the amount of suspect DNA decreases, the chance of contamination by DNA from other sources increases.